Behavioral photosensitivity of multi-color-blind medaka: enhanced response under ultraviolet light in the absence of short-wavelength-sensitive opsins

Background The behavioral photosensitivity of animals could be quantified via the optomotor response (OMR), for example, and the luminous efficiency function (the range of visible light) should largely rely on the repertoire and expression of light-absorbing proteins in the retina, i.e., the opsins. In fact, the OMR under red light was suppressed in medaka lacking the red (long-wavelength sensitive [LWS]) opsin. Results We investigated the ultraviolet (UV)- or blue-light sensitivity of medaka lacking the violet (short-wavelength sensitive 1 [SWS1]) and blue (SWS2) opsins. The sws1/sws2 double or sws1/sws2/lws triple mutants were as viable as the wild type. The remaining green (rhodopsin 2 [RH2]) or red opsins were not upregulated. Interestingly, the OMR of the double or triple mutants was equivalent or even increased under UV or blue light (λ = 350, 365, or 450 nm), which demonstrated that the rotating stripes (i.e., changes in luminance) could fully be recognized under UV light using RH2 alone. The OMR test using dichromatic stripes projected onto an RGB display consistently showed that the presence or absence of SWS1 and SWS2 did not affect the equiluminant conditions. Conclusions RH2 and LWS, but not SWS1 and SWS2, should predominantly contribute to the postreceptoral processes leading to the OMR or, possibly, to luminance detection in general, as the medium-wavelength-sensitive and LWS cones, but not the SWS cones, are responsible for luminance detection in humans. Supplementary Information The online version contains supplementary material available at 10.1186/s12868-023-00835-y.

Mammals (with the exception of monotremes) have only SWS1 and LWS in the retina.The eyes of SWS1knockout (KO) mice became electrophysiologically insensitive to UV light (λ = 360-365 nm) [16,17].Tritanope individuals lacking SWS1 exhibit a reduced luminous efficiency of violet/blue light [18].These results support an exclusive role for SWS1 in the perception of UV light or light at short wavelengths.However, such evidence in fish remains more obscure.Zebrafish with the mutated tbx2b gene exhibit differentiation of SWS1-cone precursors into rods and the lack of dispersion of melanophores in response to dorsal illumination using near-UV light [11].Acute chemical ablation of SWS1 cones in larval zebrafish reduced the sensitivity to blue and UV light but was quickly recovered within 48-72 h [19].Similar acute ablation of SWS1 cones reduced foraging performance under UV light at 1 day after the ablation in zebrafish larvae [20].An expressional switch from SWS1 to SWS2 triggered by the thyroid hormone in rainbow trout also reduced foraging performances, possibly because of the decreased UV contrast of its prey, Daphnia [21]; however, the SWS1-KO rainbow trout exhibits a malformation in the eyes and head [22].
Using medaka (Oryzias latipes or Oryzias sakaizumii), we established several types of "color-blind" strains by knocking out the cone-opsin genes for studying genotype-phenotype relationships in color vision in animals.The strain lacking LWS (LWSa and LWSb; λ max = 561 or 562 nm) did not exhibit the OMR under red light (λ ≥ 730 nm), whereas the wild-type (WT) counterpart did, up to λ = 830 nm [2,3].The lws mutant also showed a reduced body-color preference during mate choice, possibly because of a decreased ability for color discrimination [23].The strain lacking SWS2 (SWS2a and SWS2b; λ max = 439 or 405 nm) similarly exhibited a reduced body-color preference [5].However, the sws2 mutant exhibited the OMR under blue light (λ = 400 or 440 nm) as sensitively as the WT counterpart [5], likely because either 1) the absence of the blue opsin was compensated by the neighboring violet and/or green opsins, or 2) the blue opsin is not associated with the OMR.
We recently established another color-blind strain lacking SWS1 (λ max = 356 nm; [24]).Unlike the SWS1-KO rainbow trout [22], the medaka sws1 mutant was fully viable and retained the ordinary square-mosaic distribution of cones in the retina.In this study, we first focused on its behavioral phenotypes, i.e., the body-color preference under white light and the OMR under UV light.Based on the absence of apparent differences between the WT animals and the sws1 mutants (see the Results), we further established the sws1/sws2-double and sws1/sws2/ lws triple mutants and characterized their behavioral photosensitivity based on the OMR.

Mate choice of the sws1 mutant
The body colors of the color interfere (ci) mutant and the Actb-SLα:GFP transgenic strain are pale gray and dark orange, respectively [25,26].Their genomes are identical, with the exception of the transgene in Actb-SLα:GFP, which expresses a hormone (somatolactin alpha [SLα]) and Renilla green fluorescent protein (GFP) ectopically.Our previous experiments repeatedly demonstrated that these color variants mate assortatively; i.e., males strongly prefer females of the same strain [27][28][29][30].
This result (i.e., absence of a reduction in body-color preference in the sws1 mutant) was in contrast to the reduced body-color preferences of the lws [23] or sws2 [5] mutants, probably because our breeding and experiments were carried out under UV-free conditions (Fig. 1b).Although the fish may behave differently in the open air or under a light source containing UV light, the role of SWS1-expressing cones in the body-color preference seemed to be negligible under the UV-free condition.

OMR of the sws1 and sws1/sws2-double mutants under UV light
Taking advantage of the existing sws1 [24] and sws2 [5] mutants, we established a sws1/sws2-double mutant, anticipating that the potential reduction in UV sensitivity could be detected more clearly by the violet/bluedouble-color-blind fish than by the violet-color-blind fish.We crossed a sws1 −10 homozygote with a sws2 +1a+14b homozygote, which possessed 1-base and 14-base insertions in the tightly linked SWS2a and SWS2b genes, respectively [5]; raised the offspring (F 1 ), which were double-heterozygous for the sws1 −10 and sws2 +1a+14b mutations (more accurately, triple-heterozygous for the sws1 −10 , sws2a +1 , and sws2b +14 mutations); and intercrossed the F 1 to obtain F 2 individuals.
We reared the F 2 fish in the same tanks en masse (the genotypes could not be determined based on their appearance), 101 of which matured fully.Because the SWS1 and SWS2a/b loci are independent [31], the expected phenotypic ratio of WT, violet-color-blind, blue-color-blind, and violet/blue-double color-blind fish was 9:3:3:1, which was indeed observed in the F 2 generation (P = 0.385, chi-square test; Fig. 2a).Therefore, not only the sws1 or sws2 mutants [5,23], but also the sws1/ sws2-double mutant, were as viable as their WT litter mates with normal color vision, at least in our breeding conditions.
Subsequently, we investigated the OMR of the WT, sws1-mutant, and sws1/sws2-double-mutant individuals under UV light (λ = 350 or 365 nm).The spectra of the UV light for experiments are shown in Fig. 2b, together with that of the IR light for videorecording.We verified mutation.The males were given a choice between females of the Actb-SLα:GFP and ci strains, and the ratio of courtship events toward the ci females was plotted.Each dot represents one male, and the graph reports the mean value.The error bars are the 95% confidence intervals.
Regardless of the presence or absence of SWS1, the males significantly preferred Actb-SLα:GFP females, and the ratios were not statistically different between the WT and the mutant fish (P = 0.977, Student's t-test).b Normalized spectra of the white LED light used for breeding (black) and the sunlight (orange) measured at every 1 nm using a C-7000 Spectromaster (Sekonic).The colored arrows at the bottom indicate the absorption maxima (λ max ) of the medaka cone opsins [45]: SWS1 (violet); SWS2a and SWS2b (blue); RH2a, RH2b, and RH2c (green); and LWSa and LWSb (red) Fig. 2 Behavioral UV sensitivity of the sws1 or sws1/sws2 double mutants.a Establishment of the sws1/sws2 double mutants.Double heterozygotes for the sws1 −10 and sws2 +1a+14b mutations were intercrossed, and the offspring were raised under identical conditions until maturation; their genotypes (top) and phenotypes (bottom) are summarized in the tables.No significant difference was detected between the observed and expected ratios.b Normalized spectra of the UV light used for the experiments in c (blue) and d (violet).An IR spectrum of the IR camera for video recording is also shown (dark red).These spectra were measured at every 1 nm using a Sun Spectroradiometer S-2440 instrument (Soma Optics).c OMR under UV light (λ = 365 nm).Eight intensities of 0.0, 0.  that IR light alone did not induce the OMR (see the graphs at the photon flux density [PFD] of 0.0 μmol/m 2 /s in Fig. 2c, d).
At 365 nm, we examined eight PFD values, i.e., 0.00, 0.21, 0.27, 0.47, 1.44, 23.7, 76.7, and 134 μmol/m 2 /s (Fig. 2c).Importantly, all three strains (WT, the sws1 mutant, and the sws1/sws2-double mutant; n = 8 each) exhibited the OMR at a PFD ≥ 1.44 μmol/m 2 /s (note that the 95% CI did not include zero in all three strains).This result clearly demonstrated that medaka could perceive and behaviorally respond to UV light, even without SWS1 and SWS2.The OMR observed at a lower PFD was weaker (e.g., the mean of less than four rounds, and the lower limit of the 95% CI being close to zero) in all strains.Statistically, a two-way repeated-measures analysis of variance (ANOVA) detected a significant difference among the eight UV conditions (F (4.015, 80.307) = 18.723,P < 0.001, η p 2 = 0.484), but not among the three strains (F (2, 20) = 1.516,P = 0.244, η p 2 = 0.132).No interaction was detected between the UV condition and the strain (F (8.031, 80.307) = 1.118,P = 0.360, η p 2 = 0.101).That is, contrary to our expectations, the presence or absence of SWS1 and SWS2 did not significantly affect the behavioral UV sensitivity.
At 350 nm, we examined the OMR of the WT and the sws1/sws2-double-mutant individuals (n = 8 each) at five PFD values of 0.00, 5.08, 12.6, 19.5, and 25.4 μmol/ m 2 /s (Fig. 2d).We occasionally observed that the test fish uncomfortably twisted their body when the UV light was turned on.This could explain why a reverse OMR (swimming against the rotating stripes) was often observed at high UV intensities (e.g., 19.5 μmol/m 2 /s); i.e., the fish might try to escape from (rather than stay still within) the UV-light-dominated environment.Although the 95% CIs indicated that the OMR was positive at 25.4 μmol/m 2 /s in both strains (note that a few fish exhibited a nearly perfect OMR; i.e., 20 rounds), a two-way repeated-measures ANOVA did not support a significant difference among the five UV conditions (F (2.579, 30.946) = 2.065, P = 0.141, η p 2 = 0.133) or between the strains (F (1, 12) = 0.607, P = 0.451, η p 2 = 0.048).Their interaction was not significant (F (2.579, 30.946) = 0.060, P = 0.990, η p 2 = 0.005).It should be noted that all test fish were light-adapted prior to, and the rods were dysfunctional during, the OMR tests [2]; i.e., the UV light must be perceived via RH2 and/or LWS, rather than rhodopsin (RH1), in the sws1/sws2-double mutant, although other non-canonical photoreceptors could also be involved (further discussed below).

Establishment of a new OMR-testing device
In the experiments described above (Fig. 2c, d), although we paid great attention to avoiding any contamination of fluorescent light excited by UV (e.g., wrapping all the devices in aluminum foil, using vertical stripes made of strips of aluminum foil pasted on an Indian-ink-painted plastic paper, wearing gloves to avoid leaving fingerprints), there might have been some human-undetectable fluorescence that the medaka perceived and responded to.Therefore, we further investigated the behavioral photosensitivity of the sws1/sws2-double mutant in UV-free conditions.
Equiluminance (isoluminance) is an equally luminant condition between different colors, in which the recognition of differences becomes the most difficult.Hence, when the rotating stripes consisted of equiluminant colors (e.g., equally luminant green and red), the OMR should be minimized compared with that elicited by nonequiluminant colors.

Equiluminant conditions for the sws1/sws2 double mutants
Next, we changed the fixed gray color (128/128/128) to a red (255/0/0), green (0/255/0), or blue (0/0/255) color and tested the OMR of the WT and sws1/sws2-doublemutant fish (n = 8 each).We expected that, if the sensitivity to blue light was decreased in the double mutant, the blue would be equiluminant to, and therefore the OMR would be minimized in the presence of, the darker gray color in the double mutant versus the WT fish.
In the presence of red-gray stripes (Fig. 3d), the OMR was apparently positive in the extreme (i.e., red-black or red-white) conditions, thus demonstrating that these stripes were clearly visible to the WT and double-mutant fish.However, the graphs adopted a broad U shape and the condition at which the OMR was minimized was difficult to identify, particularly for the double mutant, which might have caused the significant interaction observed between the stripe conditions and the strains (F (13, 182) = 25.785,P = 0.003, η p 2 = 0.156).A two-way repeated-measures ANOVA detected significant differences among the stripe conditions (F (13, 182) = 15.181,P < 0.001, η p 2 = 0.520), but not between the strains (F (1, 14) = 0.606, P = 0.449, η p 2 = 0.042).In the presence of green-gray stripes (Fig. 3e), the OMR was minimized at a brighter gray color (190/190/190 or 200/200/200) compared with the red-gray stripes (at 140/140/140 for the WT fish).Therefore, medaka should detect the green light (0/255/0) to a greater extent than it does the red light (255/0/0), as humans do.The graphs appeared similar between the WT fish and double mutants, and no interaction was detected between the stripe conditions and strains (F (13, 182) = 0.975, P = 0.477, η p 2 = 0.065).A two-way repeated-measures ANOVA detected significant differences among the stripe conditions (F (13, 182) = 16.512,P < 0.001, η p 2 = 0.541), but not between the strains (F (1, 14) = 3.007, P = 0.105, η p 2 = 0.177).In the presence of blue-gray stripes (Fig. 3f ), the OMR was minimized at a darker gray color (100/100/100 or 110/110/110) compared with the green-gray or red-gray stripes, demonstrating that medaka detect the blue light to a lesser extent than the red or green light, similar to humans.The graph of the double mutants appeared to be flatter than that of the WT fish (as in the redgray stripes; Fig. 3d), and a significant interaction was detected between the stripe conditions and strains (F (13, 182) = 2.442, P = 0.005, η p 2 = 0.149).The dark shift of the equiluminant condition that was expected in the double mutant seemed not to occur.In fact, significant differences were detected among the stripe conditions (F (13, 182) = 17.353,P < 0.001, η p 2 = 0.553), but not between the strains (F (1, 14) = 1.278,P = 0.277, η p 2 = 0.084).Thus, the sws1/sws2 double-mutant fish seemed to sense the blue as luminant as the WT fish did.

Equiluminant red and blue for the sws1/sws2 double mutants
The "gray" color, however, consists of red, green, and "blue" light (Fig. 3b).Therefore, the reduction in bluelight sensitivity would also reduce the sensitivity to gray, which could explain why the dark shift could not be detected in the presence of blue-gray stripes (Fig. 3f ).Therefore, supposing that the lack of SWS1 and SWS2 should least affect the sensitivity to red light, we repeated the OMR test by replacing the variable gray color (0/0/0-255/255/255) with variable red color (0/0/0-255/0/0).
Taken together, neither the OMR elicited under UV (Fig. 2) nor RGB (Fig. 3) light supported the reduced behavioral UV or blue-light sensitivity in the sws1/sws2double-mutant medaka.

Establishment of the sws1/sws2/lws triple mutant medaka
To characterize further the UV perception via the green and/or red opsins, we established and analyzed a strain that possessed frameshift mutations in the SWS1, SWS2a, SWS2b, LWSa, and LWSb genes; i.e., the sws1/sws2/lws triple mutant.Because the SWS2a/b and LWSa/b loci are tightly linked on a chromosome [31], it was nearly impossible to establish the sws2/lws double mutant by crossing the existing sws2 [5] and lws [2] mutants.Therefore, we newly introduced lws mutations in the sws2 mutant (Fig. 4a, b).A total of four adult fish (G 0 ) possessed and passed the ins/del mutations in the LWSa/b genes to their offspring (F 1 ), five of which carried the double-frameshift mutations, lws −2a−1b or lws −7a+4b .Although the lws −7a+4b mutation was unfortunately lost during later crossings, we were able to establish a line that was homozygous for the sws2 +1a+14b and lws −2a−1b mutations, i.e., the sws2/lws double mutant.

Expression of the cone-opsin genes in the sws1/sws2/lws triple mutant
We considered that the color-blind mutations might increase the expression of the remaining cone opsins to compensate for the decreased repertoire (e.g., the sws1/ sws2/lws triple mutant might express the remaining RH2 more strongly compared with the WT fish).We previously found that the cone-opsin genes were differently transcribed between ci and Actb-SLα:GFP, possibly because of the ectopic expression of Renilla GFP [5].Therefore, we compared gene expression independently on the ci or Actb-SLα:GFP background using real-time reverse transcription polymerase chain reaction (RT-PCR) (Fig. 5).
On the ci background, we compared the WT (n = 3), the sws1/sws2/lws triple mutant (n = 2), and the sws1/ sws2 double mutant (n = 3) fish.An apparent reduction caused by nonsense-mediated mRNA decay (NMD) could be detected for the SWS1, SWS2a, SWS2b, and LWSa/b genes (LWSa and LWSb are 98.8% identical, and we analyzed them without discrimination) in the triple mutant and for the SWS1, SWS2a, and SWS2b genes in the double mutant (P ≤ 0.018, one-way ANOVA and posthoc Dunnett's test).By contrast, the expression of RH2a and RH2b/c (RH2b and RH2c are 95.8% identical, and we analyzed them without discrimination) was equivalent among the three strains (P = 0.891 or 0.220, respectively; one-way ANOVA).
On the Actb-SLα:GFP background, we compared the WT and the triple-mutant fish (n = 3 each).An apparent reduction triggered by NMD could be verified for the SWS1 and LWSa/b genes (P < 0.001, Student's two-tailed t-test).However, the reduction in the SWS2a or SWS2b genes was not statistically significant (P = 0.073 or 0.146, Fig. 5 Expression of the cone-opsin genes in the sws1/sws2/lws triple mutant.The expression levels of the SWS1, SWS2a, SWS2b, RH2a, RH2b/c, and LWSa/b genes in the mutant relative to those in the WT were quantified by the ΔΔCt method using the Actb gene as a reference.The RH2a and RH2b genes and the LWSa and LWSb genes were indistinguishably amplified because of similar nucleotide sequences.Top: Comparison on the ci background (the sws1/sws2 double mutant was included).Bottom: Comparison on the Actb-SLα:GFP background.Each dot represents one individual, and the graph shows the mean and the standard error.Significant differences are indicated by the P value (top: one-way ANOVA and post-hoc Dunnett's test; bottom: Student's two-tailed t-test) ▸ respectively), likely because one WT individual expressed SWS2s (and also RH2b/c) very strongly, for unknown reasons.For RH2a and RH2b/c, significant differences were not detected between the WT and the triple-mutant fish (P = 0.243 or 0.769, respectively).

Spectral sensitivity of the sws1/sws2/lws triple mutant
Lastly, we examined the spectral photosensitivity of the triple mutant via the OMR test under monochromatic light at five wavelengths (λ = 365, 450, 530, 630, or 730 nm; Fig. 6), the spectra of which are presented in Fig. 6a.We set five or six luminance conditions for each wavelength and used six fish per strain per condition; however, some fish died and needed to be replaced during the experiments, particularly at 365 nm.

Discussion
Despite our attempts to demonstrate the potential decrease in UV-or blue-light sensitivity in medaka lacking SWS1 and SWS2, none of the results presented here (Figs.2c, d, 3f-h, 6b, c) supported this assumption.In fact, the UV sensitivity might even be "increased" in the sws1/sws2/lws triple mutant (Fig. 6b).The present study should provide an important premise considering the function and evolution of cone opsins in animals; i.e., the presence or absence of a certain type of cone opsin does not necessarily affect the photosensitive behaviors of animals, even at wavelengths close to the λ max .

The OMR as an index of behavioral photosensitivity
The photosensitivity of animals could be measured using various methods (see Introduction), among which, we adopted the OMR in this and previous studies [2][3][4][5][6].The rationale was simple: when an animal does not follow the rotating stripes, it should be insensitive to the light irradiated from or reflected by the stripes.However, a more careful interpretation of the data seemed to be required.
To elicit the OMR under monochromatic light (i.e., in the condition in which all items exhibit an identical hue), animals must recognize the monochromatic stripes as a difference in luminance (brightness).In primates, the luminance is detected via the medium-wavelengthsensitive (MWS) and LWS opsins (MWS is evolutionary paralogous to LWS); moreover, the contribution of SWS, which is evolutionarily orthologous to SWS1, is restrictive [32,33].
Our present and previous results of (1) a reduced OMR under red light in the lws mutant [2][3][4] and (2) an OMR in the RH2 monochromat (the sws1/sws2/lws triple mutant; Fig. 6b-f ) demonstrated that the OMR in medaka depends on both RH2 and LWS.Alternatively, it could be considered that, rather than LWS and RH2, LWS and other non-canonical visual pigments, such as melanopsin, are responsible for the OMR, because there is a growing body of evidence showing their expression in various retinal cells [34,35] and their actual contribution to vision [36,37].In either case, SWS1 and SWS2 should play only a negligible role in the OMR at the present speed (i.e., 10 rpm), considering that the OMR of the sws1, sws2, or sws1/sws2 double mutants was not reduced at any wavelength tested in this and previous studies ( [5]; Figs.2c, d, 6b-f ).
More than a quarter of a century ago, a similar conclusion had been reached by analyzing the OMR of other fish species.Schaerer and Neumeyer [39] showed that the luminous efficiency function of goldfish (and zebrafish [40]) had a single maximum at the λ max of LWS, and therefore suggested that the LWS-expressing cones were predominantly involved in the OMR; i.e., according to those authors, the motion vision was "color-blind".A similar result was reported in cichlid [41], whereas not only LWS, but also RH2, seemed to be involved in larval zebrafish [42] and two-spotted goby [43], such as medaka.Thus, SWS1 and SWS2 would commonly be dispensable for the OMR in various fish species.Whether this is a character that is restricted to the OMR or is widely applicable to motion detection (as suggested by Schaerer and Neumeyer [38]) or luminance detection (as known in SWS of primates) warrants further investigation using methods other than the OMR test.
It should be noted that the results described above (ours and those of other researchers) only suggest the negligible role of SWS1 and SWS2 "in relation to that of RH2 and LWS"; i.e., SWS1 and SWS2 might make a significant contribution to the OMR in the absence of RH2 and LWS, and medaka that lack RH2 and LWS (the rh2/ lws double mutant) might not necessarily be OMR negative.To check this issue, we are currently knocking out three paralogs of the RH2 gene (RH2a, RH2b, and RH2c); however, the rh2 mutant seems to be less viable than its WT littermates (our unpublished observation), unlike that observed for the sws1, sws2, and lws mutants [2,5,23].This complicates the interpretation of the data, because even if the rh2 or rh2/lws double mutants exhibit a reduced OMR, this could be attributed to a reduced viability or reduced visual acuity in general, as is known in human SWS monochromats [44].
About a century ago, Schlieper [47] tested the OMR using colored and gray stripes and found conditions in which the OMR became negative, just as we did in the present study (Fig. 3d-f ).His result was initially interpreted as the tested animals being color-blind (see [40]).This interpretation was true in the sense that some fish, such as goldfish or cichlid, exhibited a "color-blind" OMR [38,40]; i.e., at the equiluminant condition, the alternating colored stripes would virtually disappear for these animals.
The OMR might also be "color-blind" for medaka (and also larval zebrafish and two-spotted goby [41,42]), in which it relies on both RH2 and LWS, because the OMR of the WT medaka similarly became negative at the equiluminant conditions (Fig. 3d-g).In Fig. 3h, however, the WT medaka consistently exhibited a positive OMR in all conditions, some of which should be equiluminant or near-equiluminant.From this point of view, it was intriguing that the sws1/sws2 double mutant generally (and statistically significantly) performed better than did the WT fish in the equiluminant conditions; i.e., there was a condition in which the OMR became negative in the WT, but not the mutant fish (e.g., Fig. 3d, f, and h).We interpreted these results as the OMR in medaka not being completely "color-blind", although the contribution of the hue (RH2-LWS opponency?; [48]) would be relatively subtle compared with that of the luminance.

Deficiency caused by the lack of SWS1
To date, we have not detected apparent morphological or behavioral defects in medaka lacking SWS1; i.e., the full viability in the laboratory ( [24]; Figs.2a, 4c), the normal cone mosaic in the retina [24], the non-reduced behavioral UV sensitivity (Fig. 2c), and the body-color preference equivalent to that of the WT (Fig. 1a).This is contrasting to the results in larval zebrafish, where acute ablation of the SWS1 cones clearly decreased the OMR and foraging performance [19,20].However, these effects in zebrafish larvae were temporal, because the ablated SWS1 cones were rapidly regenerated, which should not be argued the same way with our color-blind medaka that chronically lacks SWS1.The only phenotype we noted was the "increased" OMR in the sws1/sws2 double mutant in the equiluminant conditions (Fig. 3d, f ) and in the sws1/ sws2/lws triple mutant under UV light (Fig. 6b).Rather than transcriptional upregulation (Fig. 5), the increased UV sensitivity seemed to be achieved by other physiological (e.g., dark adaptation of the RH2-expressing cones) or morphological (e.g., retinomotor movements) mechanisms, which warrant further investigation.The series of color-blind medaka lines would be a useful model to investigate the functional relationships between cone opsin and animal behavior, which should provide an important clue for understanding the evolution of color vision in animals.

Fish
All fish were born and reared in our laboratory, where water was filtrated/circulated at 25 °C and light was provided by white LED for 14 h per day.Fish were given brine shrimps and flake foods five times per day.Sexually mature adults (more than 3 months of age) were used for all experiments.

Mate choice
A test male was given two choice females in a free-swimming condition (20 × 12 cm with a water level of 5 cm) for 30 min, and the mate preference was manually quantified as a ratio of the male's approaches.If a male was used in two or more tests, we averaged the ratios and treated this value as a single datum.We judged the preference as being significant if the 95% CI did not contain 50:50.

Genotyping
A crude extract of genomic DNA from the caudal fin was used as a template for PCR.The primer sequences used here were as follows: f: ACG CCT CTG AAC TTT GTC GTT CTT CTG and r: CTT CCA GGG CGC ACA GCG TTTG for SWS1; f: AAC AAG AAG CTT CGA TCC CA and r: ATA TCT GCA AGC GAA GGA GC for SWS2a; f: TTG TTG CTT CTA CGG GTT CC and r: TTT GGC TCT AGA GAG GTA CAG TCA for SWS2b; f: TAA ACT GGA TTT TGG TCA ATC TTG CT and r: CCA ACC ATC CTC TCA ACA GAGC for LWSa; and f: CAT AGC TGA CCT GGG AGA GACG and r: CCA ACC ATC CTC TCA ACA GAGC for LWSb (the reverse primers were identical between LWSa and LWSb).The amplified products were electrophoresed on a 12% polyacrylamide gel, and bands were detected by ethidium-bromide staining and UV irradiation (heteroduplex mobility assay [HMA]).

OMR test under monochromatic light
The diameters of a cylindrical glass tank and a rotating drum surrounding it were 9 and 15 cm, respectively.A water-filled 2-mL tube wrapped in aluminum foil was placed at the center of the glass tank to prevent shortcut during the OMR.To avoid any fluorescence under UV or blue light, vertical stripes (2-cm wide) were prepared by pasting strips of aluminum foil onto an Indian-inkpainted plastic paper, the device for rotating the drum was covered with pieces of aluminum foil, and we handled all items with gloves to avoid leaving fingerprints on the stripes or device.
Monochromatic light was provided from an LED bulb (EX-365, 450, 530, 630, or 730; Optocode) or a Max-350 xenon lamp (Asahi Spectra) with a bandpass filter of 350 nm.To adjust the intensity, we changed the output or height of the light sources and placed a reflective neutral-density filter in the light path, when necessary.PFD was directly measured using a QTM-101 quantameter (Monotech) or calculated from a spectrum measured by a S-2440 spectroradiometer (Soma Optics).For videorecording, we used an IR camera (ELP-USB100W04H-DL36-J; ELP).Its built-in IR lamps were partly covered with aluminum foil to reduce the intensity; i.e., sufficiently bright for the recording but not for the OMR.
The test fish were light-adapted under ceiling light (Additional file 1: Figure S1) for > 10 min prior to each OMR test, which consisted of a 30-s acclimation and 4 × 30-s rotations in the clockwise, anticlockwise, clockwise, and anticlockwise directions.The speed of stripe rotation was 10 rpm.We quantified the OMR as the swimming distance (rounds) in the direction of stripe rotation during the 120-s rotations.If the fish swam against this direction, the distance was added as a negative value.Therefore, the overall distance should become zero if the fish swam randomly in the dark.The positions of the test fish and the obstacle placed at the center were extracted as x-y coordinates using UMATracker software [49], and were then used for calculating the distance [3].
We calculated the mean distance and its 95% CI per strain per condition and regarded that the OMR was positive when the interval did not contain zero.For a comparison between the WT and the double/triple mutants, we performed a two-way repeated-measures ANOVA or two-way ANOVA depending on the number of fish that died during the experiments (see the Results for details) using SPSS Advanced Statistics software (IBM).

OMR test for equiluminance
The test fish were placed in the glass tank with the center obstacle (see above).A mobile display (MB16AP; Asus) was laid under the tank, and a polyvinyl-chloride mirror formatted into a conical trapezoid was placed around the tank (see Fig. 3a).The display projected sunray-shaped stripes (36 stripes with a width of 10°) spinning at 10 rpm, and the mirror horizontally reflected the image as rotating vertical stripes.The procedure used for the OMR test was identical; i.e., a 30-s acclimation and 4 × 30-s rotations.The tests were carried out under ordinary fluorescent light from the ceiling whose spectrum was provided as Additional file 1: Figure S1, the behaviors were videorecorded using the C615n webcam (Logicool), and the OMR (swimming distance) was quantified as described above.Because no fish died during these experiments, we applied a two-way repeated-measures ANOVA for statistical comparisons.

Genome editing
The detailed protocol used for knocking out the LWS genes has been described elsewhere [2,4].Briefly, the Cas9 mRNA and guide RNA targeting the 5′-GCG TGT TTG AGG GCT ATG TGG-3′ sequence of the paralogous LWSa and LWSb genes were synthesized and microinjected into embryos at the 1-cell stage.The mutations induced in the caudal fin of the injected fish (G 0 ) were detected by an HMA using the appropriate primers, and the mutated G 0 fish were backcrossed to the host strain.The mutations passed to the offspring (F 1 ) were individually sequenced, and the F 1 fish with identical double-frameshift mutations were intercrossed to obtain homozygotes.

Real-time RT-PCR
The total RNA was extracted from the eyes of adult fish using ISOGENII (Nippon Gene), contaminated DNA was digested by Doxyribonuclease (RT Grade) for Heat Stop (Nippon Gene), and cDNA was synthesized using ReverTra Ace (Toyobo) and a polyT primer.Real-time RT-PCR was carried out using the innuMIX qPCR DSGreen Standard (Analytik Jena) or the Taq Pro Universal SYBR qPCR Master Mix (Vazyme) on a qTOWER 3 G touch instrument (Analytik Jena).The thermocycling conditions were as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 20 s and 60 °C for 1 min.Primers (Table 1) were designed to sandwich the last intron of each gene and amplify products of about 150 bp.The products were relatively quantified using the ΔΔCt method with the actin beta (Actb) gene as a reference.

Fig. 1
Fig.1Mate choice of the sws1 mutant.a Body-color preference of Actb-SLα:GFP males without (black, n = 8) or with (violet, n = 16) the sws1 −10 mutation.The males were given a choice between females of the Actb-SLα:GFP and ci strains, and the ratio of courtship events toward the ci females was plotted.Each dot represents one male, and the graph reports the mean value.The error bars are the 95% confidence intervals.Regardless of the presence or absence of SWS1, the males significantly preferred Actb-SLα:GFP females, and the ratios were not statistically different between the WT and the mutant fish (P = 0.977, Student's t-test).b Normalized spectra of the white LED light used for breeding (black) and the sunlight (orange) measured at every 1 nm using a C-7000 Spectromaster (Sekonic).The colored arrows at the bottom indicate the absorption maxima (λ max ) of the medaka cone opsins[45]: SWS1 (violet); SWS2a and SWS2b (blue); RH2a, RH2b, and RH2c (green); and LWSa and LWSb (red)

Fig. 4
Fig. 4 Establishment of the sws1/sws2/lws triple mutant (i.e., the RH2 monochromat).a Genomic structure of the SWS2 (blue) and LWS (red) loci.Each locus consists of two paralogous genes (a and b).The arrows and colored boxes indicate the directions of transcription and the translated regions, respectively.The scissors indicate the approximate positions of the target sequences for CRISPR/Cas9 [2, 4, 5].b Induction of the lws mutations in the sws2 mutant.Top: Production of mosaic mutants (G 0 ) by microinjection.We obtained four G 0 adults that had the ins/del mutations in the caudal fin.Bottom: Transmission of mutations from the G 0 fish to their offspring (F 1 ).The asterisk indicates that all four F 1 fish inherited identical mutations; 2-or 1-base deletions in the LWSa and LWSb genes, respectively.c Production of the triple mutant by crossing.The sws2 +1a+14b /lws −2a−1b double mutant was crossed with the sws1 −10 mutant, and their offspring (sws1 −10 /sws2 +1a+14b /lws −2a−1b triple heterozygotes) were intercrossed.The genotypes of the mature offspring (F 2 ) are summarized in the table

Fig. 6 (
Fig. 6 (See legend on previous page.) AGC TCC TGC GTG TAC AA Reverse TTA AGA GGC CGT GGA CAC CTCCG SWS2a Forward TCA AAG GCC TCC ACT GTG TAC AAT CC Reverse CTA AGC TGG TCC GAC TTT AGA GAC TTC SWS2b Forward CCA CAG TCT ACA ACC CCT TCA TTT ATGTC Reverse TTA GGA AGG GCC GAC TTT TGA GAC TTC RH2a Forward AAA GAG CTC AGC CCT GTT CAA TCC TATC Reverse CAA GCA GCA GTA GAG ACT TCT GTC TTGC RH2b & RH2c Forward AAG AGC TCA GCA TTG TAC AAT GCT GTT ATC TA Reverse TTA AGC TGC AGT TGA GAC TTC TGT CTTGC LWSa & LWSb Forward TTT GCA AAG AGC GCC ACA ATC TAC AACC Reverse TAT GCA GGA GCC ACA GAG GAG ACC Actb Forward AGC CCT GGC CCC ATC CAC CA Reverse GAG GGG CCA GAC TCA TCG TACTC

Table 1
Primer sequences used for real-time RT-PCR